The effect of ozone on the yellowing process of magnesium-deficient clonal Norway spruce grown under defined conditions

During two vegetation periods, young clonal spruce trees (Picea abies (L.) Karst.) with sufficient and poor magnesium (Mg) supply were exposed in the environmental chambers of the GSF phytotron to three levels of ozone (daily means: 18-22, 88-130, and 135-190 microg m(-3); 10% reduction at night). Previous year's needles were examined at 4-week intervals with respect to their contents of Mg, Ca, K, Mn, N, P, and chlorophyll (Chl), various parameters of Chl fluorescence, and the stability of the isolated light-harvesting Chl-a/b-protein complex LHC II. The needles of the two nutrition variants contained more than 0.53 or less than 0.27mg Mg g(-1) needle dry matter, respectively. The ratio of variable to maximal Chl-a fluorescence of the dark-adapted needles, Fv/Fm, and the photoinhibitory quenching of Fv after light treatment, SVi.v, were affected by the Mg content of the needles rather than the ozone levels. Changes of the Chl content and the behavior of the LHC II allowed differentiating between a slow process of needle yellowing occurring under Mg deficiency only, and a rapid process of needle yellowing occurring under the combined action of Mg deficiency and ozone pollution. Only the rapid yellowing process was accompanied by destabilization of the LHC II, and the degree of destabilization was correlated with the ozone concentration present in the days before sampling. The results are consistent with observations obtained at a research site in the Central Black Forest (J Plant Physiol 161 (2004) 423).     

Expression of the skeletal muscle dystrophin-dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto-Kakizaki rat

The inability of insulin to stimulate glucose metabolism in skeletal muscle fibres is a classic characteristic of type 2 diabetes. Using the non-obese Goto-Kakizaki rat as an established animal model of this type of diabetes, sucrose gradient centrifugation studies were performed and confirmed the abnormal subcellular location of the glucose transporter GLUT4. In addition, this analysis revealed an unexpected drastic reduction in the surface membrane marker beta-dystroglycan, a dystrophin-associated glycoprotein. Based on this finding, a comprehensive immunoblotting survey was conducted which showed a dramatic decrease in the Dp427 isoform of dystrophin and the alpha/beta-dystroglycan subcomplex, but not in laminin, sarcoglycans, dystrobrevin, and excitation-contraction-relaxation cycle elements. Thus, the backbone of the trans-sarcolemmal linkage between the extracellular matrix and the actin membrane cytoskeleton might be structurally impaired in diabetic fibres. Immunohistochemical studies revealed that the reduction in the dystrophin-dystroglycan complex does not induce obvious signs of muscle pathology, and is neither universal in all fibres, nor fibre-type specific. Most importantly, the expression of alpha-syntrophin and the syntrophin-associated neuronal isoform of nitric oxide synthase, nNOS, was demonstrated to be severely reduced in diabetic fibres. The loss of the dystrophin-dystroglycan complex and the syntrophin-nNOS complex in selected fibres suggests a weakening of the sarcolemma, abnormal signalling and probably a decreased cytoprotective mechanism in diabetes. Impaired anchoring of the cortical actin cytoskeleton via dystrophin might interfere with the proper recruitment of the glucose transporter to the surface membrane, following stimulation by insulin or muscle contraction. This may, at least partially, be responsible for the insulin resistance in diabetic skeletal muscles.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

The 29.9 kDa subunit of mitochondrial complex I is involved in the enzyme active/de-active transitions

Mitochondrial respiratory chain complex I undergoes transitions from active to de-activated forms. We have investigated the phenomenon in sub-mitochondrial particles from Neurospora crassa wild-type and a null-mutant lacking the 29.9 kDa nuclear-coded subunit of complex I. Based on enzymatic activities, genetic crosses and analysis of mitochondrial proteins in sucrose gradients, we found that about one-fifth of complex I with catalytic properties similar to the wild-type enzyme is assembled in the mutant. Mutant complex I still displays active/de-active transitions, indicating that other proteins are involved in the phenomenon. However, the kinetic characteristics of complex I active/de-active transitions in nuo29.9 differ from wild-type. The spontaneous de-activation of the mutant enzyme is much slower, implicating the 29.9 kDa polypeptide in this event. We suggest that the fungal 29.9 kDa protein and its homologues in other organisms may modulate the active/de-active transitions of complex I.     

The 8.5A projection structure of the core RC-LH1-PufX dimer of Rhodobacter sphaeroides

Two-dimensional crystals of dimeric photosynthetic reaction centre-LH1-PufX complexes have been analysed by cryoelectron microscopy. The 8.5A resolution projection map extends previous analyses of complexes within native membranes to reveal the alpha-helical structure of two reaction centres and 28 LH1 alphabeta subunits within the dimer. For the first time, we have achieved sufficient resolution to suggest a possible location for the PufX transmembrane helix, the orientation of the RC and the arrangement of helices within the surrounding LH1 complex. Whereas low-resolution projections have shown an apparent break in the LH1, our current map reveals a diffuse density within this region, possibly reflecting high mobility. Within this region the separation between beta14 of one monomer and beta2 of the other monomer is approximately 6A larger than the average beta-beta spacing within LH1; we propose that this is sufficient for exchange of quinol at the RC Q(B) site. We have determined the position and orientation of the RC within the dimer, which places its Q(B) site adjacent to the putative PufX, with access to the point in LH1 that appears most easily breached. PufX appears to occupy a strategic position between the mobile alphabeta14 subunit and the Q(B) site, suggesting how the structure, possibly coupled with a flexible ring, plays a role in optimizing quinone exchange during photosynthesis.     

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.     

Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner

We have hypothesized that small RNAs may participate in learning and memory mechanisms. Because dendritic spines are important in synaptic plasticity and learning, we asked whether dicer, the rate-limiting enzyme in the formation of small RNAs, is enriched within dendritic spines. In adult mouse brain, dicer and the RNA-induced silencing complex (RISC) component eIF2c were expressed in the somatodendritic compartment of principal neurons and some interneurons in many regions, and dicer was enriched in dendritic spines and postsynaptic densities (PSDs). A portion of dicer and eIF2c were associated with each other and with fragile X mental retardation protein (FMRP), as assessed by co-immunoprecipitation. Calpain I treatment of recombinant dicer or immunopurified brain dicer caused a marked increase in RNAse III activity. Purified PSDs did not exhibit RNAse III activity, but calpain caused release of dicer from PSDs in an enzymatically active form, together with eIF2c. NMDA stimulation of hippocampal slices, or calcium treatment of synaptoneurosomes, caused a 75 kDa dicer fragment to appear in a calpain-dependent manner. The findings support a model whereby acute neuronal stimulation at excitatory synapses increases intracellular calcium, which activates calpain, which liberates dicer and eIF2c bound to PSDs. This supports the hypothesis that dicer could be involved in synaptic plasticity.     

The evolution of the human mind and logic--mathematics structures

The evolution of the human mind is discussed based on: (i) the fact that living beings interchange matter, energy and information with their environment, (ii) an ontological interpretation of the "reality" of the quantum world, of which logic-mathematics structures are considered constitutive parts, (iii) recent theories according to which living beings are considered as dynamic complex systems organized by information, and (iv) the fact that the evolution of living beings is guided by information about the environment and by intrinsic information on living systems (auto-organization). Assuming the evolution of vision as a model we observe that the driving forces that directed the evolution of the eyes, as dynamic complex systems, are the information about the environment supplied by sunlight and the intrinsic information-gaining mechanism of living organisms. Thus, there exists a convergence toward a visual system with the greatest ability to obtain light information, like the human eye, and also a divergence that leads to the development of specific qualities in some species. As in the case of vision the evolution of the human mind-brain cannot be a consequence of factors unrelated to the object of its own functioning. The human mind was structured for the acquisition from reality of the logic-mathematics structures that underlie the whole universe and consequently of an internal representation of the external world and of its own self. Thus, these structures are, together with the intrinsic capacity for auto-organization of the human brain, the predominant driving force of the human mind evolution. Both factors are complementary.